1. Field of the Invention
The present invention provides methods and compositions for treating trauma to the central nervous system (CNS). The present invention also provides methods and compositions for facilitating neuronal transplant.
2. Description of Related Art
The ACHE gene encoding the acetylcholine hydrolyzing enzyme, acetylcholinesterase (ACHE, EC 3.1.1.7), is expressed in muscle, nerve, hematopoietic cells, embryonic tissue and germ cells. ACHE maps to chromosome 7q22 and encodes the primary enzyme, acetylcholinesterase (AChE, E.C. 3.1.1.7), which terminates neurotransmission at synapses and neuromuscular junctions (NMJ). The text Human Cholinesterases and Anticholinesterases by Soreq and Zakut (Academic Press, Inc., 1993) provides a summation of the biochemical and biological background as well as the molecular biology of human cholinesterase genes. In addition Soreq et al. 1990; Seidman, et al. 1995; and Grifman et al., 1997 provide summations of various aspects of acetylcholinesterase biology. The text and references in their entirety are incorporated herein by reference.
Three alternative AChE-encoding mRNAs have been described in mammals. The dominant brain and muscle AChE found in NMJs (AChE-T) is encoded by an mRNA carrying exon E1 and the invariant coding exons E2, E3, and E4 spliced to alternative exon E6 (FIGS. 4-5). AChEmRNA bearing exons E1-4 and alternative exon ES encodes the glycolipid phosphatidylinositol (GPI)-linked form of AChE characteristic of vertebrate erythrocytes (AChE-H). An additional readthrough mRNA species retaining the intronic sequence I4 located immediately 3xe2x80x2 to exon E4 was reported in rodent bone marrow and erythroleukemic cells and in various tumor cells lines of human origin.
In addition to its classical role as the enzyme responsible for acetylcholine hydrolysis, an increasing number of studies are suggesting a non-classical role for AChE in neurogenesis [reviewed in Robertson and Yu, 1993; Layer, 1995]. This is based on observations of intense patterns of AChE activity occurring transiently in many developing neural structures before synaptogenesis, or in locations which have no cholinergic synapses. In the vertebrate retina, four AChE-positive subbands have been described in the IPL [Marc, 1986; Hutchins, 1987], only two of which correspond to ChAT-positive subbands [Millar et al., 1985]. The other two are apparently not associated with cholinergic transmission. One possible explanation for these non-cholinergic AChE subbands is that they are related to neurite guidance. Several studies have demonstrated non-catalytic functions of AChE in the regulation of neurite outgrowth from embryonic neurons [Layer et al., 1992; Layer et al., 1993; Small et al., 1995].
It is proposed that AChE, and other cholinesterase-like molecules, are involved in cell-cell recognition. AChE displays homology to nervous system adhesion proteins such as neurotactin [de la Escalera, 1990; Darboux et al., 1996], gliotactin [Auld et al., 1995], and neuroligin [Ichtchenko et al., 1995]. Moreover, certain isoforms of AChE may possess an HNK-1 epitope that is commonly found on cell adhesion proteins [Bon et al., 1987].
Closed head injury (CHI) is a major cause of mortality and morbidity among young adults and an important risk factor in non familial Alzheimer""s disease [French et al., 1991; Gentleman et al., 1993; Mayeux et al., 1995]. Following head trauma, disruption of the blood-brain-barrier contributes to the development of vasogenic edema. In addition, release of autodestructive factors leads to cytotoxicity and acute as well as delayed neuromotor and cognitive impairments [Caprusi and Levine, 1992; Hamm et al., 1996; Gennarelli, 1997]. The early phase of post-injury responses also includes a burst of released acetylcholine [Gorman et al., 1989] and elevated levels of intracellular calcium [Siesjo, 1993] in the brain. Pre-injury administration of the muscarinic antagonist scopolamine facilitates recovery from brain injury [Hamm et al., 1993], suggesting that rapid suppression of the early immediate intense stimulation mediated by acetylcholine released cholinergic hyperexcitation, during the first few post-injury minutes post-injury is therapeutically advantageous. However, other methods are needed that intervene at biologically significant steps so that recovery is assured and long-term risk factors for neurodegenerative diseases are avoided.
Acute cholinergic stimulation itself promotes a rapid and prolonged elevated overproduction (overexpression) of AChE [Friedman et al., 1996]. These elevated levels of AChE will act to brake the immediate phase of cholinergic hyperactivation. However, protracted overexpression of AChE will excessively suppress cholinergic neurotransmission.
Apart from its catalytic role, accumulated evidence establishes non-catalytic, neurite growth-promoting activities for AChE [Layer and Willbold, 1995, Koenigsberger et al., 1997; Sternfeld et al., 1998]. This suggests that elevated AChE levels might also promote a secondary phase of dendritic hypertrophy that could be important for short-term recovery from head injury such as CHI. Yet, it has been recently observed that extended overexpression of neuronal AChE in brain and spinal cord of transgenic mice promotes reduced dendritic branching, loss of dendritic spines (i.e. less synapses), and delayed, neuromotor and cognitive deficits [Beeri et al., 1995, 1997; Andres et al., 1997]. Together, these observations therefore raised the possibility that acute cholinergic stimulation following head trauma promotes an upregulation of AChE biosynthesis that is beneficial in the short term, but which causes long-term perturbations in the normal dendritic reorganization that takes place in the adult brain [Flood and Coleman, 1990, Arendt et al., 1995]. If so, the increased risk of AD among survivors of severe head injuries could be viewed as a delayed consequence of too long an exposure to AChE following injury and can potentially create imbalanced neurite extension and impaired targeting due to the neurite guidance role of AChE as described herein above. It would therefore be useful following head injury and any other injury to the central nervous system (CNS) to insure that an excess of AChE does not interfere with recovery, i.e. that a balance of AChE levels and timing is maintained. This is particularly critical in those patients which are already compromised in that their neural AChE levels are elevated due to biological, genetic or environmental factors.
It has been recently demonstrated [Chen et al, 1997A] that treatment of CHI with a brain specific inhibitor of anticholinesterase catalytic activity had a positive effect on short term recovery. However exposure to AChE enzymatic inhibitors itself activates a feedback loop leading to elevated levels of mRNA for AChE (AChEmRNA) [Friedman et al., 1996] and therefore this treatment has a potential for long term overexpression of AChE and subsequent development of neurodegenerative disease.
As with any therapy an appropriate model is required, either in vivo, ex vivo or in vitro. Since mice do not naturally overexpress AChE, Applicants have generated a unique transgenic mouse model for Alzheimer""s Disease to serve this purpose [Beeri et al., 1995]. These genetically engineered mice overproduce human AChE in cholinergic brain cells providing a model of overexpressed AChE. Applicants"" transgenic mice display age-dependent deterioration in cognitive performance as initially measured by a standardized swimming test for spatial learning and memory and a social recognition test. Since the excess acetylcholinesterase in the brains of these mice is derived from human DNA, it is a model for any intervention directed against human acetylcholinesterase protein and/or gene. This animal system and brain slices derived thereof, therefore provide the ability to test therapies by in vivo, ex vivo and in vitro means to restore balanced cholinergic signaling in the brain.
Transplantation of neural tissue into the mammalian CNS is a potential therapeutic treatment for neurological and neurodegenerative disorders including epilepsy, stroke, Huntington""s diseases, head injury, spinal injury, pain, Parkinson""s disease, myelin deficiencies, neuromuscular disorders, neurological pain, amyotrophic lateral sclerosis, Alzheimer""s disease, and affective disorders of the brain. For example, fetal ventral mesencephalic tissue has been demonstrated to be a viable graft source in Parkinson""s disease. [Lindvall et al., 1987; 1990; Bjorklund, 1992]. Likewise, fetal striatal tissue has been utilized successfully as graft material in Huntington""s disease [Isacson et al., 1986; Sanberg et al., 1994].
Neurologically dysfunctional animals have been transplanted with non-fetal, non-neuronal cells/tissue. The major advantage of this type of transplantation protocol is that the graft source is not a fetal source and, thereby, circumvents the ethical and logistical problems associated with acquiring fetal tissue [Bjorklund and Stenevi, 1985; Lindvall et al., 1987]. It would be useful to be able to also use neural grafts of non-fetal neuronal cells and to improve the graft integration (form connections) with the CNS of the recipient (i.e. the host) as has been shown by Wictorin et al. [1990].
According to the present invention, a method of treating injury to the central nervous system (CNS) is provided. The method includes administering to the CNS of a patient suffering from such an injury a therapeutically effective amount of an inhibitor of acetylcholinesterase production immediately following the injury. The treatment downregulates acetylcholinesterase production and thereby activity. The injury to the CNS may be a head injury (closed or open) or a spinal cord trauma or other trauma to the CNS.
The method uses as the inhibitor of acetylcholinesterase production a synthetic nuclease resistant antisense oligodeoxynucleotide or a ribozyme wherein they are directed against an accessible domain of the AChEmRNA brain variant and pharmaceutical compositions thereof. In an embodiment the inhibitor is at least one synthetic nuclease resistant antisense oligodeoxynucleotide selected from 5xe2x80x2ACGCTTTCTTGAGGC 3xe2x80x2 (SEQ ID No:1),
5xe2x80x2GGCACCCTGGGCAGC 3xe2x80x2 (SEQ ID No:2),
5xe2x80x2CCACGTCCTCCTGCACCGTC 3xe2x80x2 (SEQ ID No:3),
5xe2x80x2ATGAACTCGATCTCGTAGCC 3xe2x80x2 (SEQ ID No:4),
5xe2x80x2GCCAGAGGAGGAGGAGAAGG 3xe2x80x2 (SEQ ID No:5),
5xe2x80x2TAGCGTCTACCACCCCTGAC 3xe2x80x2 (SEQ ID No:6),
5xe2x80x2TCTGTGTTATAGCCCAGCCC 3xe2x80x2 (SEQ ID No:7), and
5xe2x80x2GGCCTGTAACAGTTTATTT 3xe2x80x2 (SEQ ID No:8).
The method further includes the inhibitor being administered a second time following monitoring of the patient and determining upon magnetic resonance imaging (MRI), that c-fos activity is still seen.
The present invention further provides a method of facilitating transplantation of neuronal cells to the CNS by administering to the patient a therapeutically effective amount the acetylcholinesterase inhibitor of production or pharmaceutical composition thereof at the time of transplant. The neuronal cells to be transplanted can be neurons of fetal origin, neurons of adult origin or a neuronal cell line and can be genetically modified to produce a noncatalytic brain specific variant (E6) of acetylcholinesterase under control of an inducible promoter.
The present invention also provides a method of improving hippocampal neuron survival following injury to the central nervous system by administering to a patient suffering from such an injury a therapeutically effective amount of an inhibitor of acetylcholinesterase production to the central nervous system of the patient immediately following the injury. The injury to the central nervous system may be a closed or open head injury or a spinal cord trauma.
The method uses as the inhibitor of acetylcholinesterase production a synthetic nuclease resistant antisense oligodeoxynucleotide or a ribozyme directed against an accessible domain of the AChEmRNA brain variant or a pharmaceutical composition thereof. In an embodiment the inhibitor is at least one synthetic nuclease resistant antisense oligodeoxynucleotide selected from 5xe2x80x2ACGCTTTCTTGAGGC 3xe2x80x2 (SEQ ID No:1),
5xe2x80x2GGCACCCTGGGCAGC 3xe2x80x2 (SEQ ID No:2),
5xe2x80x2CCACGTCCTCCTGCACCGTC 3xe2x80x2 (SEQ ID No:3),
5xe2x80x2ATGAACTCGATCTCGTAGCC 3xe2x80x2 (SEQ ID No:4),
5xe2x80x2GCCAGAGGAGGAGGAGAAGG 3xe2x80x2 (SEQ ID No:5),
5xe2x80x2TAGCGTCTACCACCCCTGAC 3xe2x80x2 (SEQ ID No:6),
5xe2x80x2TCTGTGTTATAGCCCAGCCC 3xe2x80x2 (SEQ ID No:7), and
5xe2x80x2GGCCTGTAACAGTTTATTT 3xe2x80x2 (SEQ ID No:8).
The present invention provides a pharmaceutical or medical composition for the treatment of injury to the central nervous system comprising as active ingredient at least one inhibitor of acetylcholinesterase in a physiologically acceptable carrier or diluent. The active ingredient can be a synthetic nuclease resistant antisense oligodeoxynucleotide or a ribozyme directed against an accessible domain of the AChEmRNA brain variant or a combination thereof.
In an embodiment the synthetic nuclease resistant antisense oligodeoxynucleotides are selected from the group consisting of 5xe2x80x2ACGCTTTCTTGAGGC 3xe2x80x2 (SEQ ID No:1),
5xe2x80x2GGCACCCTGGGCAGC 3xe2x80x2 (SEQ ID No:2)
5xe2x80x2CCACGTCCTCCTGCACCGTC 3xe2x80x2 (SEQ ID No:3),
5xe2x80x2ATGAACTCGATCTCGTAGCC 3xe2x80x2 (SEQ ID No:4),
5xe2x80x2GCCAGAGGAGGAGGAGAAGG 3xe2x80x2 (SEQ ID No:5),
5xe2x80x2TAGCGTCTACCACCCCTGAC 3xe2x80x2 (SEQ ID No:6),
5xe2x80x2TCTGTGTTATAGCCCAGCCC 3xe2x80x2 (SEQ ID No:7), and
5xe2x80x2GGCCTGTAACAGTTTATTT 3xe2x80x2 (SEQ ID No:8).